1. Field of the Invention
This invention relates to an antiserum, antibody, and IgG fraction which are directed against an antigen that is a substantially nonfluorescent dye, and which upon mixing with the dye turn it fluorescent.
2. Related Background Art
Spectroscopic analysis is conventionally in frequent use for analyzing a minute amount of substances in biological metabolites, or contaminants in the environment. Especially, absorption spectrum analysis and fluorescence spectrum analysis are widely employed because their methods of measurement are simple.
Although the absorption spectrum analysis has lower sensitivity in detection as compared with the fluorescence spectrum analysis (hereinafter referred to as xe2x80x9cfluorescence analysisxe2x80x9d) which will be described in detail below, it has been more widely utilized because the subject substances to be detected often possess light absorption in the ultraviolet-visible region.
In absorption spectrum analysis, absorbance and a concentration of the subject substance to be detected are expressed according to the following equation:
Absorbance=log(I0/I)=xcex5cl
where I0 is the intensity of incident light, I is the intensity of light that has transmitted through a sample, xcex5 is the molar absorption coefficient of a subject substance to be detected, and l is an optical path length.
As the equation shows, the measurement of absorbance provides for the ratio of intensity of the incident light to that of the transmitted light, which does not vary with an increase in the intensity of a light source or in the sensitivity of a detector.
In contrast, if the subject substance to be detected is fluorescent, fluorescence analysis may be utilized. In this case, since the quantity of light to be emitted by the subject substance when it is exposed to excitation light is measured, this enables the sensitivity of measurement to be improved by employing a stronger light source or increasing the sensitivity of the detector.
Specifically, the intensity (F) of light emitted as fluorescence, the concentration (c) of a fluorescent substance, and the intensity (I0) of excitation light (or incident light) are correlated with each other, and the intensity (F) is proportional to the intensity (I0) of the incident light as follows:
F="PHgr"I0(1xe2x88x9210xe2x88x92xcex5cl)
where I0 is the intensity of incident light, xcex5 is the molar absorption coefficient of a subject substance to be detected, l is an optical path length, and "PHgr" is a fluorescence quantum yield.
In cases where the subject substance to be detected is included in a sample, which is a mixture comprised of many components, it often happens that the absorption spectra of the contaminants may overlap those of the subject substance during the measurement of absorbances. This results in difficulty selectively measuring only the absorbance of the subject substance.
On the other hand, even in the case where the subject substance to be detected is included in a sample, which is a mixture comprised of many components, in fluorescence analysis, contaminants that are not fluorescent can be ignored. It is possible to selectively measure only the fluorescence of the subject substance by choosing the excitation and fluorescence wavelengths when fluorescent contaminants are coexistent in the sample.
For these reasons, fluorescence analysis is superior to absorption spectrum analysis in terms of the sensitivity in detection, as well as, of the possibility of measuring a mixture.
Therefore, in an analytical technique where the advantage that fluorescence analysis allows a high-sensitivity measurement, is combined with the high selectivity of an antigen-antibody reaction, antibodies directed against fluorescent dyes are conveniently prepared. When these antibodies are reacted with the dyes, they either decrease or increase the fluorescence intensities of the dyes.
Accordingly, based on the variations in fluorescence intensity, quantitative analysis specific to these dyes have been carried out.
However, there has been an inherent limitation to the high sensitivity, highly selective fluorescence analysis employing the aforementioned antigen-antibody reaction in that it is not applicable in the case where the subject substance to be detected is substantially nonfluorescent.
It is also known that a dye which is substantially nonfluorescent under normal conditions of measurement may turn fluorescent (i.e., acquire fluorescent ability) under specific conditions, thereby enabling fluorescent measurement.
For example, while Malachite Green (hereinafter referred to as xe2x80x9cMGxe2x80x9d) is normally nonfluorescent, it is known to turn fluorescent in glycerin. There has been an explanation concerning this phenomenon that the rotations in a molecule of the dye is restricted under the influence of a solvent or the like and as a result, the dye molecule turns fluorescent.
Many fluorescent substances do exist in vivo and the fluorescence of these substances (autofluorescence) forms a background in the measurement of fluorescence of a biological sample, which may result in a decline in the sensitivity of detection. Also, in the measurement of immunofluorescence (hereinafter referred to as xe2x80x9cfluoroimmunoassayxe2x80x9d) employing titer plates, fluorescence emitted by plastic materials of the titer plate similarly forms a background, which may result in a decline in the sensitivity of detection. These are the causes that preclude fluorescence measurement with high sensitivity in the prior art.
In consideration of the foregoing, an object of this invention is to provide an antibody capable of enhancing or manifesting its fluorescent ability by specifically binding to a dye which is substantially nonfluorescent under normal conditions of measurement and by restricting the rotations in a molecule of the dye owing to the binding. Also, an object of this invention is to provide an antibody capable of binding to a dye that enables the measurement with higher sensitivities as compared with other dyes because of its extremely low background fluorescence.
The invention provides an antiserum which comprises an antibody directed against an antigen having a substantially nonfluorescent dye, wherein a mixture of the antiserum or antibody and the dye is provided with fluorescent ability.
Further, the invention provides an IgG fraction which comprises a fraction derived from an antiserum containing an antibody directed against an antigen that has a substantially nonfluorescent dye, wherein a mixture of the IgG fraction and the dye is provided with fluorescent ability.
Also, the invention provides the antiserum as described above wherein the antigen is formed by conjugation of an immunogenic substance with the dye.
In addition, the invention provides the IgG fraction as described above wherein the antigen is formed by conjugation of an immunogenic substance with the dye.
Furthermore, the invention provides the antiserum as described above wherein the immunogenic substance is at least one member selected from the group consisting of bovine serum albumin, human serum albumin, egg albumin, bovine xcex3 globulin, equine serum globulin, human xcex3 globulin, ovine xcex3 globulin, bovine thyroglobulin, porcine thyroglobulin, hemocyanin, and a synthetic polypeptide.
Also, the invention provides the IgG fraction as described above wherein the immunogenic substance is at least one member selected from the group consisting of bovine serum albumin, human serum albumin, egg albumin, bovine xcex3 globulin, equine serum globulin, human y globulin, ovine xcex3 globulin, bovine thyroglobulin, porcine thyroglobulin, hemocyanin, and a synthetic polypeptide.
Still further, the invention provides the antiserum as described above wherein the dye has a triphenylmethane moiety.
Also, the invention provides the IgG fraction as described above wherein the dye has a triphenylmethane moiety.
Also, the invention provides the antiserum as described above wherein the dye having the triphenylmethane moiety is Malachite Green.
Also, the invention provides the IgG fraction as described above wherein the dye having the triphenylmethane moiety is Malachite Green.
Further, the invention provides the antiserum as described above wherein the dye has a diphenylmethane moiety.
Also, the invention provides the IgG fraction as described above wherein the dye has a diphenylmethane moiety. Also, the invention provides the antiserum as described above wherein the dye having the diphenylmethane moiety is Auramine O.
Also, the invention provides the IgG fraction as described above wherein the dye having the diphenylmethane moiety is Auramine O.
According to the invention, an antibody directed against an antigen which is a substantially nonfluorescent dye is prepared and the antibody and the dye are mixed, thereby allowing the substantially nonfluorescent dye to turn fluorescent and enabling the fluorescence analysis of the dye in minute quantities. Further, the use of MG in the fluorescence measurement can reduce the background level to a great extent as compared with the use of other dyes such as CCJV (9-(2-carboxy-2-cyanovinyl)julolidine) or fluorescein, enabling higher sensitivity in measurement.
The present invention will be more fully understood from the detailed description given hereinbelow and the accompanying drawings, which are given by way of illustration only and are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.